ACAA2 Knockout HeLa Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal population of HeLa cells engineered to disrupt the ACAA2 gene, which encodes the mitochondrial 3-ketoacyl-CoA thiolase enzyme. This product provides a genetically defined loss-of-function model for investigating the terminal step of fatty acid ??-oxidation without the need for clonal isolation. The polyclonal format reflects a heterogeneous cell pool harboring diverse CRISPR-mediated disruptive mutations across the ACAA2 locus, enabling robust population-level studies of metabolic perturbations while maintaining the practical advantages of a pooled knockout system.
HeLa cells, derived from an HPV-18 positive human cervical adenocarcinoma, are an immortalized epithelial line extensively employed in cancer biology and cell signaling research. Their transformed phenotype, characterized by rapid proliferation and altered metabolic dependencies, makes them particularly suitable for examining lipid catabolism and metabolic reprogramming. The HeLa background provides a well-characterized genomic and metabolic context, facilitating the integration of ACAA2 knockout data with decades of existing literature on tumor cell metabolism, drug responses, and mitochondrial function.
ACAA2 encodes a thiolase that catalyzes the thiolytic cleavage of 3-ketoacyl-CoA into acetyl-CoA and a shortened acyl-CoA, executing the final reaction of the mitochondrial ??-oxidation spiral. This enzyme functions downstream of the long-chain acyl-CoA dehydrogenase ACADVL and the trifunctional protein subunits HADHA and HADHB, and in close coordination with ECHS1. ACAA2 activity is transcriptionally regulated by PPAR?? and PGC-1??, and is modulated by energy-sensing kinases such as AMPK and hormonal signals from glucagon. The generated acetyl-CoA enters the TCA cycle, contributes to ketone body synthesis via HMGCS2, or generates reducing equivalents for ATP production, thereby linking lipid catabolism to core bioenergetic pathways. Cofactors including CoA, NAD+, and FAD are essential for the upstream and downstream reactions, while CPT1A and CACT control the entry of fatty acyl chains into the mitochondria.
Within the HeLa host cell, ACAA2 disruption allows dissection of the reliance on mitochondrial ??-oxidation for energy production and anabolic support. Because tumor cells often rewire metabolic networks, this knockout model can reveal whether HeLa cells compensate for defective fatty acid oxidation through enhanced glycolysis or glutaminolysis, providing insights into metabolic flexibility. The interplay between PPAR??-mediated transcriptional programs and the physical loss of ACAA2 activity creates a controlled system to study the consequences of ACAA2 deficiency, which is associated with metabolic acidosis and potential cancer metabolic dysfunction. This polyclonal knockout model therefore serves as a relevant in vitro surrogate for pathological states involving impaired lipid oxidation.
Researchers can employ this product to measure fatty acid oxidation flux using radiolabeled or stable isotope-labeled substrates, monitor acyl-CoA species accumulation via LC-MS metabolomics, and assess mitochondrial respiration with Seahorse respirometry under lipid-dependent conditions. RT-qPCR and western blotting confirm ACAA2 disruption and evaluate compensatory changes in PPAR?? targets or ketogenic enzymes. Viability assays in galactose or lipid-supplemented media further probe metabolic vulnerabilities. The model supports drug screening efforts targeting lipid metabolism and studies of metabolic reprogramming in cancer. For further technical details, please contact Ascent Research.
